Pseudomonas aeruginosa, and this enzyme orchestrates several physiopathological events during bacteria-host interplays. LasB is considered to be a potential target for the development of an innovative chemotherapeutic approach, especially against multidrug-resistant strains. Recently, our group showed that 1,10-phenanthroline-5,6-dione (phendione), [Ag(phendione)2]ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) had anti-P. aeruginosa action against both planktonic- and biofilm-growing cells. In the present work, we have evaluated the effects of these compounds on the (i) interaction with the lasB active site using in silico approaches, (ii) lasB proteolytic activity by using a specific fluorogenic peptide substrate, (iii) lasB gene expression by real time-polymerase chain reaction, (iv) lasB protein secretion by immunoblotting, (v) ability to block the damages induced by lasB on a monolayer of lung epithelial cells, and (vi) survivability of Galleria mellonella larvae after being challenged with purified lasB and lasB-rich bacterial secretions. Molecular docking analyses revealed that phendione and its Ag+ and Cu2+ complexes were able to interact with the amino acids forming the active site of lasB, particularly Cu-phendione which exhibited the most favorable interaction energy parameters. Additionally, the test compounds were effective inhibitors of lasB activity, blocking the in vitro cleavage of the peptide substrate, aminobenzyl-Ala-Gly-Leu-Ala-p-nitrobenzylamide, with Cu-phendione having the best inhibitory action (Ki = 90 nM). Treating living bacteria with a sub-inhibitory concentration  of the test compounds caused a significant reduction in the expression of the lasB gene as well as its mature protein production/secretion. Further, Ag-phendione and Cu-phendione offered protective action for lung epithelial cells, reducing the A549 monolayer damage by approximately 32 and 42%, respectively. Interestingly, Cu-phendione mitigated the toxic effect of both purified lasB molecules and lasB-containing bacterial secretions in the in vivo model, increasing the survival time of G. mellonella larvae. Collectively, these data reinforce the concept of lasB being a veritable therapeutic target and phendione-based compounds (mainly Cu-phendione) being prospective anti-virulence drugs against P. aeruginosa.Elastase B (lasB) is a multifunctional metalloenzyme secreted by the gram-negative pathogen  Pseudomonas aeruginosa infections is particularly challenging due to the increasing global prevalence of MDR strains  strains . The fai strains . This gr strains . Also, Pelements . Accordiectively .Pseudomonas aeruginosa has a large range of extracellular virulence attributes , which are responsible for increasing its pathogenicity  protease, whose synthesis is guided by the expression of the lasB gene and is regulated by the quorum sensing (QS) transcription systems, las and rhl  of host cells, inducing tissue injury and hemorrhage  is the most abundant protease harvested from pseudomonal secretome, and it has been extensively studied . LasB is and rhl . LasB ismorrhage . Also, lmorrhage  by degramorrhage . Corroboceration . Likewise strain .P. aeruginosa, the development of innovative chemotherapy is of immediate necessity  interaction with the amino acids forming the lasB active site, (ii) lasB proteolytic activity, (iii) lasB gene expression, (iv) lasB protein secretion, (v) ability to block the damages induced by lasB on a lung epithelial cell line, and (vi) survival of Galleria mellonella larvae challenged with purified lasB molecules and lasB-containing bacterial secretions.Faced with the upcoming post-antibiotic era, the anti-virulence approach emerges as a promising strategy against superbug infections as it acts by disarming the virulence arsenal of the pathogen and, consequently, reducing the ability of the organism to damage host cells . Therefoosa lasB . Herein,2] ClO4 (Ag-phendione) and [Cu(phendione)3](ClO4)2.4H2O (Cu-phendione) , , and (ii) calculate their hydrogen bonding interaction energies with amino acid residues within the cavity of the lasB enzyme .In this theoretical analysis, docking calculations were employed in order to (i) identify favorable structural features of phendione, Ag-phendione and Cu-phendione through calculation of interaction energies with the known P. aeruginosa lasB crystallographic structure containing the HPI inhibitor  (®)   by using4 Å) (®) , taking 4 Å) (®) . For theThe MolDock scoring function employed in the MVD program comes from the piecewise linear potential (PLP), a simplified potential whose parameters are fitted to protein-ligand structures, binding data scoring functions and further extended in the Generic Evolutionary Method for molecular docking with a new hydrogen binding term and new charge schemes . Next, EWherein:PLP  consists of two different parameter sets: one for the approximation of the steric term  among the atoms, and the other potential for the hydrogen binding. The second term is related to the electrostatic interactions among overloaded atoms. It is a Coulomb potential with a dielectric constant dependent on the distance (D(r) = 4r). The numerical value of 332.0 is assigned for the electrostatic energy  :El mol–1) . Eintra θ is the torsional angle of the bond. If several torsions could be determined, each torsional energy is considered and the average value used. The last term, Eclash, assigns a penalty of 1,000 if the distance between two heavy atoms (more than two bonds apart) is smaller than 2.0 Å, disregarding infeasible ligand conformations (The first part of the equation (double summation) is among all pairs of atoms in the ligand, taking off those that are connected by two bonds. The second one characterizes the torsional energy, wherein rmations . The docrmations .In this stage, a further theoretical investigation was carried out in order to evaluate the electronic effects in obtaining the interaction energy of the phendione-based test compounds. The semi-empirical calculations were based on methods developed by Michael Polanyi and Henry Eyring in 1931 , for couThe application of these techniques is quite important in order to acquire a better understanding of the possible interaction modes of the test compounds within the lasB active cavity.P. aeruginosa strains were used in the experiments: a reference strain (ATCC 27853), which was formerly isolated from blood  for 18 h at 37°C. Subsequently, planktonic bacterial cells were subcultured in tryptone soy broth (TSB) supplemented with 1% glycerol, 50 mM glutamate, 10 mM CaCl2 and 10 mM ZnCl2 and incubated for 24 h at 37°C under constant agitation (8 colony-forming units/mL) in TSB medium. Next, 100 μL of the culture was pipetted into each well of a flat-bottomed 96-well polystyrene plate and incubated for 24 h at 37°C. Subsequently, biofilm supernatants were collected, combined in a single tube and filtered through a 0.22 μm membrane.gitation . CultureThe cell-free supernatants were concentrated 10-fold in a 10,000 molecular weight cut-off Centricon micropartition system . The protein concentration was determined using the method described by p-nitrobenzylamide , bonded to the elastase of P. aeruginosa. The reaction mixtures (100 μL) containing the purified lasB of P. aeruginosa  at 10 ng per 100 μL/well or cell-free supernatants (20 μg of protein), the peptide substrate (200 μM) and the reaction buffer  were incubated in the absence or in the presence of different concentrations of phendione, Ag-phendione, Cu-phendione and simple salts (AgNO3 and CuSO4.5H2O). The fluorescence was monitored over 20 min at 30 s intervals using a SpectraMax Gemini XPS Fluorescence Microplate Reader  at an excitation wavelength of 330 ± 10 nm and an emission wavelength of 460 ± 10 nm. The inhibitory constant (Ki) values were calculated using a derivation of the Michaelis–Menten equation employing GraphPad Prism 6.1. The Michaelis–Menten constant (Km) value for the substrate, required for the calculation, was experimentally determined as 13.2 μM using a Lineweaver–Burk Plot.The activity of secreted lasB was measured using a microtiter-based assay as described by 2, 1 mM ZnCl2 and 150 mM NaCl, pH 8.0 (The protease profile was assayed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) containing 0.1% gelatin incorporated into the gel as proteinaceous substrate . Gels we, pH 8.0 . The gelP. aeruginosa cells with phendione-based compounds, bacterial cells (106) were cultured in TSB medium for 24 h at 37°C with shaking in the absence and in the presence of each compound at the concentration corresponding to the ½ × MIC value, as previously established by our group ].In order to investigate the impact on lasB production by treating living ur group : phendioP. aeruginosa cells .The detection of lasB extracellularly released into the culture supernatant was performed using a Western blotting assay . Brieflysa cells . Based osa cells . The hom® bacterial RNA isolation kit  in accordance with the manufacturer’s instructions. The concentration of RNA was measured using Nanodrop 2000 (Thermo Fisher Scientific) and the quality was evaluated by resolving on a 1.5% agarose gel at 120 V. Real-time one-step PCR was performed in a 96-well plate on the purified RNA samples according to the QuantiFast® SYBR® Green RT-PCR Handbook protocol . Five microliters of both forward and reverse primers (1 μM) and 1 μl RNA (∼100 ng) were added to each well . The primers used were lasB  and the housekeeping gene rpoD (forward: 5′-GGGCGAAGAAGGAAATGGTC-3′ and reverse: 5′-CAGGTGGCGTAGGTGGAGAA-3′)  . As prevAGAA-3′) , the Rpoed cells . To do tpression .2 cell culture flasks containing Dulbecco’s modified Eagle medium  supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in an atmosphere of 5% CO2. For the experiments, A549 cells were seeded (104 per well) into 96- or 24-well plates containing the above medium and grown to confluence. Firstly, 50 μL of the cell-free pseudomonal supernatant  and purified lasB (10 ng protein) were previously treated with 25 μM of each phendione-based compound for 1 h at 37°C. Then, 20 μL each of the neutralized supernatant and the neutralized purified lasB were added to the A549 monolayer in 180 μL of fresh DMEM, followed by incubation for 48 h at 37°C in an atmosphere of 5% CO2. In parallel, A549 cells exposed only to phendione-based compounds were used as a viability control. Cell viability was determined by the reduction of the 3--2,5-diphenyl-2H-tetrazolium (MTT) bromide salt, and the integrity of the monolayer as well as the cell morphology were observed using light-field microscopy  cells were maintained in 75 cmcroscopy .G. mellonella were obtained at the insectarium of Biochemistry Department, Chemistry Institute from UFRJ, Brazil. Breeding and maintenance of G. mellonella larvae were carried out as previously described (P. aeruginosa strains (ATCC 27853 and 09HC) were obtained, filtered and concentrated as described above. Then, groups of 10 larvae were inoculated with 10 μL of the (i) pseudomonal supernatants (containing 100 μg of protein), (ii) thermal-inactivated supernatants (100°C for 5 min) and (iii) purified LasB molecules (1 μg) by directly injection into the hemocoel via the last left pro-leg, using a 25 μL Hamilton syringe. As a physical injury control, the same volume of PBS was also inoculated into G. mellonella larvae. In addition, an extra control group of larvae without any inoculation were employed to evaluate the health of larvae during the follow-up of the experimental conditions. After injection, the larvae were incubated in glass dishes without food and in the dark at 37°C for up to 7 days. Survival was appraised daily at the same time as the beginning of the experiment, and the larvae were considered as being dead when no reaction was observed upon stimulus  and Dunnett’s multiple comparison test using GraphPad Prism 6 computer software. In –1), followed by phendione  and Ag-phendione  (198 and Asn112) in the lasB catalytic site, as well as two other hydrogen bonds with water molecules (113) and three water molecules  . Corroboolecules . Likewisolecules , while p–1), followed by Ag-phendione  and phendione  (–1) and a parallel trend was also observed from the docking studies. Based on these findings, it is evident that phendione and its metal-based complexes could actively interact with lasB. Given that Cu-phendione presented the best fit to the HPI/lasB active site model and also had the greatest interaction energy, this metal complex presents the most favorable attributes for interaction with the P. aeruginosa lasB catalytic site.In addition, semi-empirical calculations were performed at PM6 level in order to obtain more precise values of their hydrogen bonding interaction energies with amino acid residues within the cavity of the lasB enzyme, and comparing these new findings with those obtained by the docking analyses. In agreement with the docking data, Cu-phendione presented the greatest relative interaction energy  . The intin silico analyses, the in vitro impact of the phendione compounds on the enzymatic activity of lasB was investigated using a specific fluorogenic peptide substrate ], followed by Ag-phendione (Ki = 0.31 μM) and phendione (Ki = 0.38 μM)  . The simactivity .P. aeruginosa cells (P. aeruginosa cells (ATCC 27853 and 09HC)  . Cu-phennd 09HC) . Furthernd 09HC) .lasB gene expression, as well as the production of its mature protein product, the lasB secreted form. To do this task, live P. aeruginosa cells were subjected to sub-inhibitory concentrations  of the test compounds for a 24 h period. Phendione and its metal-containing complexes down-regulated the lasB gene expression in a similar way, reducing it by approximately 50% compared with the untreated cells    and disronectin) . Thus, ionectin)  and induonectin) . In addial cells . In contal cells . Furtheral cells . Likewisal cells . Finallyin vitro lasB enzymatic activity, the efficacy of Cu-phendione (the most potent lasB inhibitor) on the attenuation of the toxicity of pseudomonal supernatant was explored in an in vivo infection model using the larvae of the insect, G. mellonella. Firstly, the protective ability of Cu-phendione was tested against the in vivo toxicity mediated by the supernatants obtained from the ATCC 27853 and 09HC strains , with the ATCC strain producing around 1.4-times more lasB than the 09HC strain  for 30 min and then injected into the larvae. It was found that the P. aeruginosa supernatant was noxious to G. mellonella larvae in a typical dose-dependent manner (G. mellonella larvae (G. mellonella larvae in concentrations up to 50 μM (data not shown).Since the ATCC 27853-derived supernatant was more toxic toward t manner . Interesa larvae . In addia larvae . Cu-phenP. aeruginosa is a constant threat in relation to community- and hospital-acquired infections, primarily because of the emergence and quick spread of refractory strains that are unresponsive to every clinically available antimicrobial drug , and thus the addition of metal-chelator molecules (ligands) with the capability of abstracting the central metal ion from the protein are expected to inhibit its enzymatic activity. Accordingly, multidentate EDTA and EGTA prevent lasB enzymatic activity by sequestering both Zn2+ and Ca2+ ions that are essential for its proteolytic activity and its three-dimensional structure, respectively  P. aeruginosa clinical strains recovered from the sputum of patients suffering from various airway infections , repressed lasB gene expression in P. aeruginosa by 84% (i) values of 0.31 and 0.09 μM, respectively. Additionally, treating P. aeruginosa cells with sub-inhibitory concentrations of the phendione-based compounds repressed the expression of the lasB gene as well as preventing the production and/or secretion of mature lasB protein. The molecular mechanisms underlying reduced lasB gene expression and other virulence factors in P. aeruginosa is due to the QS regulatory networks. It is well known that QS regulatory networks respond to environmental stress cues , and the two main regulatory networks are las and rhl gene systems that activate the expression of QS-responsive genes (and proposed to constitute nearly 10% of the P. aeruginosa genome). However, more recent studies have reported the existence of two additional QS networks (quinolone-based and integrated QS) that are integrated and interconnected with the las and rhl systems  (P. aeruginosa PAO1 strain (The invasiveness of  of lasB . LasB pr of lasB . Consequ8 μg/mL) . It was 8 μg/mL) . Diethyl1 strain . Moreove1 strain . The prein vivo model could provide more robust results that may help more in the understanding of the biological effects of drug treatment . Those authors correlated a lower virulence profile of antibiotic-resistant P. aeruginosa strains with their notably poor lasB production. Since the ATCC 27853 supernatant was more toxigenic to G. mellonella larvae, we selected this strain to further investigate the in vivo anti-virulence effects of Cu-phendione. It was observed that larvae inoculated with pseudomonal supernatant exhibited lower survival rates when compared to the larvae that were injected with a Cu-phendione-neutralized supernatant. P. aeruginosa pathogenesis, the inhibition of this enzyme could possibly suppress the successful establishment of the bacterial infection. Previous studies have revealed that the deletion of lasB gene culminates in a less invasive infection in rabbit corneal epithelial cells  block the production of secreted lasB. Additionally, due to their ability to inhibit lasB, these compounds nullified the lasB cytotoxic effects on lung epithelial cells. Interestingly, earlier in vivo experiments demonstrated that Ag-phendione and Cu-phendione are well tolerated by G. mellonella larvae and Swiss mice  interact with the active site of lasB, (ii) decrease the activity of purified lasB and secreted lasB obtained from bacterial supernatants, (iii) inhibit the iss mice . Importaiss mice . CorroboThe raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.All authors conceived and designed the experiments. AG, LV, AC, EC, TM, LM, MO, and TR performed the experiments. All authors analyzed the data. MP, MH, OH, MD, MM, TR, MB, and AS contributed to reagents, materials, and analysis tools. All authors wrote and revised the manuscript, contributed to the research and approved the final version of the manuscript, and agreed to be accountable for all aspects of the work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Here, we present a miR mechanism which is active in the nucleus and is essential for the production of intron included, C-terminal truncated and biologically active proteins, like e.g. Vim3. We exemplified this mechanism by miRs, miR-15a and miR-498, which are overexpressed in clear cell renal carcinoma or oncocytoma.Both miRs directly interact with DNA in an intronic region, leading to transcriptional stop, and therefore repress the full length version of the pre-mRNA, resulting in intron included truncated proteins (Mxi-2 and Vim3). A computational survey shows that this miR:DNA interactions mechanism may be generally involved in regulating the human transcriptome, with putative interaction sites in intronic regions for over 1000 genes.In this work, an entirely new mechanism is revealed how miRs can repress full length protein translation, resulting in C-terminal truncated proteins. In these times of personalized medicine, the detection of new biomarkers for the identification, differentiation or early diagnosis of diseases is of great importance.Recently we identified a structural protein, called Vimentin variant 3 (Vim3), which is the only known histological biomarker usable for the differentiation of oncocytoma (benign kidney cancer) and eosinophilic variant of the clear cell renal carcinoma (eRCC). The clear differentiation between oncocytoma and malignant kidney tumor types will be essential in the future for patients’ therapy as well as operative planning, follow-up therapy, and patients’ survival .The identification of Vim3 as a biomarker was initiated by discrepant results of different commercially available antibodies for Vimentin: One directed against epitopes in the C-terminus of the full length Vimentin (V9), the other against epitopes in the rod domain (exon 4–5) of the full length version (3B4). For the V9 antibody, most or nearly all oncocytoma were negative and all RCCs were positive. In contrast, using the 3B4 antibody both tumor types were positive. We found that a published, truncated splice variant of Vimentin  was able to explain why these two different antibodies give such discrepant results. Surprisingly, in 2011 Thakkar described the presence of this truncated variant in gliomas [By comparing the amino acid sequences of Vimentin and Vim3 it is noticeable that both are homolog until exon 7, where the Vim3 variant is truncated and ends up with an additional C-terminal ending of 8 amino acids. Interestingly, a biomarker for RCC, Mxi-2, is a truncated version of MAPK14 that follows the same mechanism (patent pending).et al. [Besides proteins, micro RNAs (miRs) are a promising candidate for biomarkers. As an example, we have identified miR-15a to be overexpressed in RCCs tissue  and has et al. .In general miRs are short (21-23 nucleotides), non-coding RNAs that play important roles in cell function and development by targeting mRNA sequences of protein-coding transcripts –7. This et al. demonstrate the localization of miRs in the nucleus and their association with Ago2 [In the cytoplasm the Ago2 complex is involved in the interaction of miRs with the target RNA sequences , 10. Furith Ago2 . They deith Ago2 . So far,ith Ago2 .et al. published in 2012 that in mouse the translocation of miRs into the nucleus is responsible for the post-transcriptional regulation of pri-miRs [It has been assumed that the nucleus is a possible storage portal for miRs which release from the nucleus in case of e.g. cell stress . Tang etpri-miRs . Furtherpri-miRs . A similpri-miRs .Investigating our tumor collective for mutual miRs for the differentiation of benign and malignant kidney cancer as biomarkers, we identify miR-498 which is overexpressed in oncocytoma and miR-15a which is known to be overexpressed in RCCs tissue as well as in the urine.We hypothesized that there is a possible relationship between miR translocation into the nucleus and an increased expression of C-terminal truncated, intron included biologically active proteins. Therefore, we compared the C-terminal sequences of both truncated proteins with both miRs of possible interaction sites.In this work, we identify miR-498 as a biomarker for oncocytoma. We show that it, as well as the biomarker miR-15a, interacts with DNA in the nucleus leading to an isoform switch of Vimentin and MAPK14. Interestingly, this partially intron retention does not lead to nonsense mediated decay, but the resulting C-terminally truncated proteins, Mxi-2 and VIM-3, are biologically active. Furthermore, computational analysis shows that there are many more putative intronic targets of known micro RNAs. Thus, this is probably a common mode of regulation through miRs.This newly identified mechanism of miR function opens a new field for the biological research area, as the function of most of these identified C-terminally truncated proteins is unknown and increased therefore the portfolio of our known proteins.In 2012 we showed that miR-15a is a biomarker for the differentiation between oncocytoma and malignant variants of kidney cancer in patient’s urine . Re-analBased on these data, we hypothesized that the two miRs (mir-15a and miR-498) were responsible for the “truncation” reaction on the DNA level. While the presence of miRs in the nucleus, where they may be involved in different mechanisms like e.g. gene expression regulation has already been described earlier , 14, 19,To this end, we needed to show translocation from the cytoplasm into the nucleus. To visualise the translocation of miRs in the nucleus, we transfected 293T cells with a FITC-labelled miR-15a and FITC-labelled miR-498 and treated them with Endothelin-1 (ET-1), since ET-1 is responsible for the translocalisation of miRs into the nucleus  and performed immunoprecipitation of these marked miRs. After this, DNA isolation and PCR with specific target gene primers showed that an interaction between the miRs and the DNA takes place Figure  and 5C. 98 and peOur results and the calculated energy values show that the interaction between miRs and DNA is feasible.To show that miR:DNA interaction leads to splicing-terminal truncation, we designed a double fluorescence vector with a green fluorescence protein at the C-terminus and a blue fluorescence protein at the N-terminus. Between these fluorescence proteins the multiple cloning site is located, into which either the Mxi-2 or the Vim3 sequence was cloned. Every positively transfected cell shows a green and a blue fluorescence signal. The blue signal disappears in case of an interaction between the miRs and the vector Figure  and 5D. p < 0.0001) of very stable interactions when using an approximate energy model [p-value < 0.01, a minimum number of target sites of 20, and at least one target site with a binding energy <−30 kcal/mol are reported here  is believed to be essentially based upon the presence of a 3′ polyA tail deadenylation , either In conclusion, miRs can bind to the non-coding DNA strand and induce a transcriptional stop that results in the production of proteins with truncated C-termini. Computational experiments suggest that this mode of miR function might be used much more often than in the two cases shown here. If this is the case, this can have a profound influence on the human protein repertoire, with many putative truncated isoforms that may have influence in many diseases.www.DSMZ.de, P/N ACC 635) were ccultured in Dulbecco’s Modified Eagle Medium , supplemented with 10% FCS  and 1% Penicillin/Streptomycin  at 37° C and 5% CO2. For all experiments, cells were pre-incubated for 24 h with serum-free DMEM.293T cells (Cells were treated with endothelin-1 (ET-1) (50 nM) for different time points. The cells were serum starved for 24 h before treatments.Before performing the immunoprecipitation, cells were transfected with FITC-labeled miR oligos, either with or without mutation and then stimulated with ET-1. For FITC-miR-immunoprecipitation, an anti-FITC mouse antibody  was incubated with an A/G PLUS-agarose antibody (Santa Cruz) on ice for 2 h. Afterwards the whole DNA, isolated either from treated cells or appropriate controls, was incubated on ice under the same conditions as described below. The samples were washed twice with 1xPBS and a PCR was performed. As negative control only agarose beads, an unlabeled miR and FITC-labeled mutated miR were used. Half of the PCR product was loaded on a 2% agarose gel and the other half was used for Sanger sequencing.Ago2 antibody  was used. Cell fractions were separated according to the nuclear extraction protocol. The different fractions were incubated for 1 h on ice with the Ago2 antibody, followed by an incubation period on ice for 2 h with A/G PLUS-agarose antibody (Santa Cruz). Afterwards either the DNA (Mxi-2 detection/PCR) or the miR was isolated and PCR was performed.® V3.1 Sequencing Kit  protocol. The product was sequenced by the Cologne Center for Genomics and analysed with the 4Peaks V1.7.2  and BLAST [The PCR product from the FITC-miR- immunoprecipitation was prepared for Sanger sequencing according to the BigDyend BLAST .The QIAamp DNA mini kit  was used for DNA extraction of transfected 293T cells, according to the manufacturer’s protocol.The digest was performed before FITC-miR-immunoprecipitation, using the New England Biolabs DNaseI (RNase-free) enzyme  according to the provided protocol.The RNeasy FFPE kit  was used for miR isolation of the FFPE samples, according to the manufacturer’s protocol. RNA quantification was accomplished using the NanoDrop technology . The miR was isolated from cells with the miRNeasy Kit , according to the manufacturer’s protocol.A Nikon F601 camera  connected to an Olympus microscope DP71  was used for fluorescence imaging. The CellP^2.5 Program, Olympus soft imaging solutions (Olympus) was used to evaluate the fluorescence pictures.AseI and BamHI-HF (New England Biolabs). The digested product of the pEGFP sized ∼1350 bp was cloned in the resulting ∼4200 bp pEBFP. The final product was a green/blue vector with a multiple cloning site (MCS) between both signals. The green/blue vector was digested with XhoI and KpnI-HF (New England Biolabs) and the PCR amplified, XhoI and KpnI-HF digested Mxi-2 product  was cloned (pEGFP/Mxi-2/EBFP). Furthermore, two mutated versions of this vector were also generated (pEGFP/Mxi-2 gc mut/EBFP and pEGFP/Mxi-2 atggtaat mut/EBFP). Analog to pEGFP/Mxi-2/EBFP the vector pEGFP/VIM3/EBFP was constructed. Cells were either transfected with: miR-15a oligo alone (100 µM), the pEGFP/EBFP vector (1 µg), pEGFP/Mxi-2/EBFP (1 µg) or pEGFP/Mxi-2/EBFP plus miR-15a oligo (1 µg + 100 µM). Cells were analysed with the Cell^P program (Olympus) 24 h and 48 h after transfection. All experiments were performed with four different clones.Subconfluent cells were grown in a monolayer on 6 well plates. 100 nM oligos were always used for transfection. The oligo was diluted in 100 µl OptiPAN  containing 6 µl FuGENE HD . The transfection was performed according to the FuGENE HD protocol. The whole suspension was drop-wise re-suspended in 900 µl OptiPAN medium including 50 nM ET-1. After 1 and 3 h the samples were analyzed and DNA was isolated as described in DNA extraction. A double-labelled vector was constructed for pEGFP/EBFP analysis. The commercially available vectors pEGFP and pEBFP  were digested with Nuclear and cytoplasmic extracts were isolated from treated cells or controls according to the manufacturer’s protocol . Protein content was assayed with the Bradford protein assay , with bovine serum albumin as standard.Formalin-fixed and paraffin embedded (FFPE) human samples from the archives of the Department of Pathology, University Hospital of Cologne, Germany were used. Histologic evaluation was based upon analyses by a staff pathologist (J.W.U. Fries) using Hematoxilin-Eosin stained paraffin-sections.Since human materials  were used (12 and 6 per group), procedures were followed as outlined in accordance with ethical standards formulated in the Helsinki Declaration 1975, with pre-approval by the local Ethics Committee at the University Hospital (reference number: 09-232).The cDNA was obtained from 250 ng RNA using random primers and SuperScript III reverse transcriptase according to the manufacturer’s protocol . The RT-PCR was performed as previously described , 25.The RNeasy FFPE kit from Qiagen was used for miRNA isolation of the FFPE samples, according to the manufacturer’s protocol. RNA quantification was accomplished using the NanoDrop technology. The miRNA was isolated from cells with the miRNeasy Kit from Qiagen, according to the manufacturer’s protocol.T method was used for calculation as outlined in User Bulletin 2 . Untreated cells were used as controls. For the statistical significance of qRT-PCR values student’s t-test was applied.1 µl of the cDNA (transcribed from 250 ng RNA) was used for real-time PCR analysis. The experimental settings were as previously described , 25. ForThese values were calculated as followed: the difference between 1 and 3h ET-1 treated and untreated cells were calculated first, the cytoplasmic levels were used as baseline levels and calculated against the values resulting from the NE fraction. Afterwards the miR-15a or the miR-498 was compared between 1 and 3 h. In both cases a statistical significant increase was detectable.t-test was utilized. The ImageJ [t-test was applied for statistical analysis with the result that all differences between cytoplasmic (cyto) vs nuclear extracts (NE) have a statistical significance .All experiments were performed in a set of three independent experiments. The GraphPrism 5  program was employed for the statistical analysis. For statistics a Student’s paired e ImageJ  lab versWestern blot analysis was performed as previously described , 25. ForWe used GENCODE19 annotation  of the h


Kaempferia galanga (KG) using an activity-guided approach. KG rhizomes were serially extracted with petroleum ether, chloroform, methanol and water. These extracts (2 g/kg each) were tested for their ability to inhibit carrageenan-induced rat paw edema. The chloroform extract was found to exert the highest inhibition (42.9%) compared to control (p < 0.001), hence it was further fractionated by washing serially with hexane, hexane-chloroform (1:1) and chloroform. The chloroform fraction (1 g/kg) showed the highest inhibitory effect  on carrageenan-induced edema. This chloroform fraction was further fractionated with hexane-chloroform (1:3) and chloroform, and of the two fractions, the hexane-chloroform sub-fraction was the most effective in inhibiting edema . GC-MS analysis of the active sub-fraction identified ethyl-p-methoxycinnamate (EPMC) as the major component, which was re-crystallized. EPMC dose-dependently inhibited carrageenan-induced edema with an MIC of 100 mg/kg. Moreover, in an in vitro study, EPMC non-selectively inhibited the activities of cyclooxygenases 1 and 2, with IC50 values of 1.12 µM and 0.83 µM respectively. These results validate the anti-inflammatory activity of KG which may be exerted by the inhibition of cyclooxygenases 1 and 2. EPMC isolated from this plant may be the active anti-inflammatory agent. This study evaluated the anti-inflammatory effect of  Inflammation can be simply defined as a response of tissue to cell injury. It may be involved in almost every pathological condition that causes cell injury or necrosis. That is why inflammation is a common concern in a number of diseases, ranging from minor insect bites to much more complicated and serious conditions like cancer. It is newly been evidenced from serological studies of different biomarkers as well as by recently emerging imaging technologies that chronic vascular inflammation is involved not only in atherosclerosis, but also in other related conditions such as arterial hypertention and metabolic syndrome . ChronicKaempferia galanga Linn. (Zingiberaceae) is indigenous to tropical Asia, where it is commonly used in traditional medicine for the management of swelling, rheumatism, cough, dysentery, diarrhea, and stomachache .Male Sprague Dawely (SD) rats (150–200 gram) were obtained from the animal research and service Centre (ARSC), Universiti Sains Malaysia. The animals were kept in the animal transit room, School of Pharmaceutical Sciences, Universiti Sains Malaysia . The animals were provided free access to water and food K. galanga. The extracts were reconstituted in 1% Tween 80 in distilled water; hence it was administered to group 5 which formed the control group. After this single dose extract administration, signs of possible toxicity were observed every hour for the first 6 h and every day for 14 days. All animals were weighed daily and observed for any signs or symptoms of toxicity and/or mortality for up to 14 days. Food and water consumption were recorded daily. The visual observations included changes in the skin and fur, eyes and mucous membranes, and also in the respiratory, circulatory, autonomic and central nervous system as well as somatomotor activity and behavioral pattern. A similar evaluation of EPMC toxicity was done later in the experiment. The animals were divided randomly into two groups of six rats each. Group 1 animals were orally given a single limit dose  of EPMC reconstituted in 1% tween 80 in distilled water. Group 2 animals were given 1% Tween 80 in distilled water. The animals were monitored for 14 days as mentioned above. In both acute toxicity studies, the animals were euthanized on the last day of experiment and LD50 value was estimated.Healthy adult female SD rats (200–225 g) were used in the acute toxicity study. The study was conducted according to the fixed dose procedures  . The ratK. galanga extracts. However, after fractionation of the most active crude extract, the dose of the resultant fractions F-1, F-2 and F-3 used in the bioassay procedure was reduced by 50%, to 1 gram/kg. Likewise, the dose of the sub-fractions SF-1 and SF-2 was also reduced by 50%, to 500 mg/kg. EPMC isolated from the most effective sub-fraction was administered in graded doses of 100 mg/kg, 200 mg/kg, 400 mg/kg and 800 mg/kg. 1% Tween 80 in distilled water was used throughout the experiment to reconstitute the extracts. Indomethacin at 5 mg/kg (a dose simulated from human regimen) was used as reference drug at every stage of the experiment [A maximum dose of 2 g/kg was arbitrarily used for the preliminary assessment of anti-inflammatory effect of crude periment , while 1K. galanga crude extracts, fractions, and sub-fractions was assessed by carrageenan-induced rat hind paw edema as described by Samud and co-workers [ad libitum. Rats were divided into groups of six animals each (n = 6). In vivo assay of anti-inflammatory effect was carried out in four stages, viz: with the crude extracts , fractions , sub-fractions (sub-fractions 1 and 2) and isolated EPMC. At each stage, treatment groups consisted of rats given extracts/fraction/sub-fraction/EPMC, reference drug indomethacin (positive control) and 1% tween 80 in distilled water (negative control). Exactly 1 h after this oral treatment, 0.1 mL of 1% freshly prepared carrageenan in normal saline was injected into the sub-plantar region of right hind paw. The thickness of right hind paw of rats was then measured using micrometer before (baseline) and after 1st, 2nd and 3rd h of carrageenan administration. The % inflammation was calculated using following formula:Anti-inflammatory effect of -workers  with minwhere A = Pad thickness, 3 h after carrageenan-induced edema; B = Pad thickness before carrageenan-induced edema.Cyclooxygenase inhibitory effect of EPMC was assessed using cyclooxygenase (COX) inhibitory screening assay kits. Briefly, COX-1 (ovine) and COX-2 (human recombinant) were incubated separately with test samples  for 15 min in test tubes and arachidonic acid was added. The reaction mixture was incubated at 37 °C in water bath for 2 min. This was followed by the addition of diluted hydrochloric acid and saturated stannous chloride solution into the reaction mixture of each test tube to stop the reaction. After an incubation of 18 h, the reaction mixture was taken on mouse anti-rabbit IgG coated micro-plate. Prostaglandin antiserum and later a prostaglandin tracer (prostaglandin antibodies linked with acetyl cholinesterase) were added to the wells of the micro-plate. After several washings, Ellman’s reagent was added to the wells and the absorbance of light was recorded in a micro plate reader at a wave length range of 405 to 420 nm.p < 0.05, p < 0.01, and p < 0.001.Data obtained from animal experiment were expressed as the mean ± standard error (±SEM). Statistical difference between the treatments and the control were evaluated by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Differences were considered significant at K. galanga possesses significant anti-inflammatory effect; hence the study contributes towards validation of the traditional use of K. galanga in the treatment of inflammation. Ethyl-p-methoxycinnamate isolated from K. galanga is found to be the vital anti-inflammatory constituent that exerts its anti-inflammatory effect via inhibition of the activities of cyclooxygenase enzymes 1 and 2.Our results indicate that


This study investigates the role of language background and bilingual status in the perception of foreign lexical tones. Eight groups of participants, consisting of children of 6 and 8 years from one of four language background (tone or non-tone) × bilingual status —Thai monolingual, English monolingual, English-Thai bilingual, and English-Arabic bilingual were trained to perceive the four Mandarin lexical tones. Half the children in each of these eight groups were given auditory-only (AO) training and half auditory-visual (AV) training. In each group Mandarin tone identification was tested before and after (pre- and post-) training with both auditory-only test (ao-test) and auditory-visual test (av test). The effect of training on Mandarin tone identification was minimal for 6-year-olds. On the other hand, 8-year-olds, particularly those with tone language experience showed greater pre- to post-training improvement, and this was best indexed by ao-test trials. Bilingual vs. monolingual background did not facilitate overall improvement due to training, but it did modulate the efficacy of the Training mode: for bilinguals both AO and AV training, and especially AO, resulted in performance gain; but for monolinguals training was most effective with AV stimuli. Again this effect was best indexed by ao-test trials. These results suggest that tone language experience, be it monolingual or bilingual, is a strong predictor of learning unfamiliar tones; that monolinguals learn best from AV training trials and bilinguals from AO training trials; and that there is no metalinguistic advantage due to bilingualism in learning to perceive lexical tones. Like consonants and vowels, lexical tone is subject to perceptual attunement as a product of specific language experience. However, unlike consonants and vowels, lexical tone is not used to distinguish meaning in all the languages of the world. While tone languages comprise 70% of the world's languages Yip,  and moreLexical tone is a linguistic device contributing to the semantic realization of words. The main cue for lexical tone is fundamental frequency, perceived as pitch, but lexical tone is also characterized by variations in amplitude, duration and voice quality Yip, . Tones vTones 2 and 3 are the most difficult to perceive for first language (L1) children and second language (L2) adults. For example, Wong et al.  found thDifficulties in discrimination of non-native language sounds by L2 tone language learners would appear to be related to perceptual attunement in infancy. Newborn infants perceive both native and non-native speech contrasts in a similar manner, but this language-general speech perception becomes more language-specific over infants' first year—there is a decline in discrimination performance for non-native speech contrasts while that for native speech contrasts is maintained or improves  school children in First Grade (6–7 years old) and Third Grade (8–9 years old) for their perception of non-native lexical tones.Auditory training improves non-native tone perception in adult populations. For example, Wang et al.  tested AIn another study, tone language Mandarin Chinese and non-tonal English speakers were trained to perceive Cantonese Chinese lexical tones ; the other bilingual group with one non-tone, English, and one tone, Thai, language background (Bi-Eng/Thai). In addition, there were two groups of monolingual children – one non-tonal, English (Mono-Eng) and one tonal (Mono-Thai).The study reported here is the first to focus on the intricacies of training non-native lexical tone perception to monolingual vs. bilingual children with or without tone language experience. Four groups of primary (elementary) school students in two age groups, First grade (6–7 year-olds) and Third grade (8–9 year-olds), were trained  to perceive non-native, Mandarin, lexical tone contrasts. Two of the four groups were bilingual: one bilingual group with two non-tonal language backgrounds: English and Arabic  or Auditory-Visual (AV) computer-based 4-alternative forced choice-identification tasks across six training sessions.Since tone language experience has been shown to facilitate lexical tone perception, it is expected that children with tone language experience would be better able to perceive foreign lexical tones, and those with a non-tone language background will have difficulty perceiving tones. Thus perception accuracy and improvement over training on Mandarin tones is expected to be better for children with tone language experience (Bi-Eng/Thai and Mono-Thai groups) than those without (Bi-Eng/Arabic and Mono-Eng groups).Moreover, it is expected that bilinguals (Bi-Eng/Thai and Bi-Eng/Arabic) should show better performance than monolinguals (Mono-Thai and Mono-Eng) due to greater metalinguistic awareness that comes with the ability to attend to and transfer across languages.In addition, as there has been found to be visual augmentation of auditory tone perception in adults, it is expected that groups of children given Auditory-Visual training will perform better than those given Auditory-Only training, although this is proposed tentatively, as visual perception of tone has not yet been studied in children.Finally, while there is only two years between the two age groups here, 6 and 8 years, it is possible that the reduced ability to perceive non-native speech contrasts in the second period of perceptual attunement around reading onset may affect the younger, 6-year-old, more than the older 8-year-old children.As there has been found to be a relation between children's speech perception and reading and reading-related abilities Burnham,  and betwA sample of 81 primary school students participated in this study. The children were either bilingual (Bi-Eng/Thai or Bi-Eng/Arabic) or monolingual (Mono-Thai or Mono-Eng) and they had either a non-tone (Bi-Eng/Arabic or Mono-Eng) or tone (Bi-Eng/Thai or Mono-Thai) language background. Within each language group, there were two age groups, First Grade 6 to 7 years [6yo] and Third Grade 8–9 years [8yo], and within each language × age sub-group children were randomly assigned to either an Auditory-Only (AO) or an Auditory-Visual (AV) training group, prior to and irrespective of their scores on the Pre-training tests.Mage = 8.07, SD = 1.15) – 12 6yo ; 12 8yo .Bi-Eng/Arabic: 24 children  – 12 6yo ; 12 8yo .Bi-Eng/Thai: 24 children  – 8 6yo  – 8 6yo ; 8 8yo .Mono-Thai: 16 children  at least one parent was a native speaker of Thai or Arabic, depending on the group, and spoke to their child in that language on a daily basis; (b) the children systematically learned both languages either at their normal school or at a language school; and (c) the parents reported that their children used each language on a daily basis. Parents or guardians also filled out a questionnaire regarding their child's language and musical training background. Only one child in the Bi-Eng/Arabic and three in the mono-Eng groups had received musical training while about 50% of the Bi-Eng/Thai group were engaged in Thai musical training at the time of testing as a requirement of the Thai language Saturday school curriculum. The study was conducted under the Western Sydney University and the University of Kansas Human Research Ethics Committees' approval in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki).The study employed a mixed, 2 × 2 × 2 × 2 × (2 × 2) factorial design: 2 Language Background groups  and 2 Tone Language Experience groups  × 2 Age groups (6yo or 8yo) × 2 Training Modes  between-subject factors × 2 Test Types  × 2 Test Phases (Pre-/Post-training) within-subject factors. The main dependent variable for accuracy was the percentage of correct tones identified. Participants in each Language × Age group were randomly assigned to either the AO or AV Training in equal numbers for each group. The order of the Test trials  was counterbalanced between participants within groups. In addition, the three of the four groups who had English as (one of) their language(s) were also given phonological awareness tests, including a phoneme deletion and a reading ability (words and non-words) test.Stimuli used in the experiment were auditory-visual recordings of monosyllabic Mandarin syllables from six native speakers of Beijing Mandarin Chinese . One female speaker (Speaker F3) provided stimulus items for the introductory session and the practice test. Another female speaker (Speaker F4) provided stimulus items for the Pre- and Post-training Test Phase stimuli. Four other speakers, two female  and two male  provided stimulus items for the training sessions.www.adobe.com). Sound was sampled at a frequency of 48,000 Hz. Target syllables were presented stationary in the center of a computer screen one at a time. Two separate files were then created from the same recording session for each stimulus, one for the Auditory-Only (AO) training and another for the Auditory-Visual (AV) training condition. Note that for the AO, a still image of the speaker was presented.The recordings were conducted in a soundproof booth in the Face and Voice Lab at MARCS Institute for Brain, Behavior and Development, Western Sydney University. The speakers were asked to produce target syllables via an AKG lapel microphone connected to an SONY HD video camera. The recording sessions were recorded via Adobe Premiere Pro CS5 program (ha/), spoken with each of the four Mandarin tones, as well as one repetition of the resultant 12 trials.A total of 12 stimuli were created for the introductory session. These comprised Speaker F3 productions of 3 syllables  with the four Mandarin tones . Another set of 24 stimuli were created for the practice session. These comprised Speaker F3 productions of 3 syllables , 24 for each of the four Mandarin tones were created for the pre-training and post-training tests. These comprised speaker F4 producing 24 syllables on each of the four Mandarin tones. The stimuli were identical in the pre-training and post-training tests but were re-randomized in each test. These 24 syllables were .A total of 144 stimuli (words and non-words), not present in the pre-training and post-training tests or practice were created for the six training sessions  for all participants irrespective of training (AO or AV) mode; a practice session (with AO or AV training depending on the training group): and the first training session (AO or AV). The second to fifth sessions consisted of only a training session  and the sixth session consisted of AO or AV training plus post-training tests . Feedback  was given in practice and in AO or AV training sessions but not in the pre-training or post-training ao and av tests. In all training sessions, participants were also rewarded with a short cartoon clip every time they made three consecutive correct responses. The six sessions were scheduled at two sessions per week over 3 weeks. In the third session, the three groups with English as one of their languages  also completed the three phonological awareness tasks, Phoneme Deletion, and Word reading and Non-word reading.Testing and training were conducted in quiet classrooms at each school; or at MARCS BabyLab, Western Sydney University Bankstown Campus; or at public library near the participant's home. A maximum of four children were tested at one time, using four identical laptops and associated hardware and software.The results are presented in three parts: (a) a comparison of total raw accuracy in pre-training vs. post-training tests, irrespective of Training Mode (AO/AV) and test mode (ao/av) in an Age (6 vs. 8 years) × Language Background  × Tone Language Experience  × (Mean Test Score – Pre-/Post-Training) design with repeated measures on Pre- vs. Post-Training scores; (b) an analysis of a percentage gain due to training dependent variable derived from the Pre- and Post-Test scores (see formula below) in an Age × Language Background × Tone Language Experience × Training Mode (AO/AV) × (ao vs. av Tests) design, with repeated measures on ao vs. av tests; and (c) a set of correlations between the phoneme deletion and word and non-word reading ability tests and with the pre- and post-tests and gain due to training for the three English-speaking groups  for whom data on the phonological awareness and reading tests was collected.F = 3.898).Raw percentage correct data were first analyzed to show the absolute level of performance Post-Training compared to Pre-Training as a product of the group factors. A 2 × 2 × 2 × (2) Analysis of Variance (ANOVA) was conducted with Age, Language Background and Tone Experience as between-subject factors and Phase (Pre- vs. Post-training test), as the within-subject factor. All factors have two levels so no planned contrasts were required. Alpha was set at 0.05 and the effect sizes are given for significant differences  = 7.61, p < 0.01, ηp2 = 0.077, with Post-Training Mean = 28.43, and SD = 0.09, and Pre-Training Mean = 25.76, and SD = 0.06.The results are graphically presented in Figure F = 9.90, p < 0.005, ηp2 = 0.395, and a Phase × Age × Tone Experience, F = 15.40, p < 0.001, ηp2 = 0.505, interaction. As can be seen in Figure As can be seen in Figure decrease in performance from pre- to post-training by the monolingual tone language (Thai) 6-year-olds is puzzling. These children had just begun instruction in reading and writing at school, including learning the orthographic representation of Thai tones . This raises the issue of the degree of improvement given the initial level of performance and the equivalence of improvements from an initial level of chance responding vs. a higher level of initial responding. To accommodate such differences a dependent variable was derived as follows:Thus if a child had 20% correct on Pre-Training and 30% on Post-Training—the Performance Gain would be 50%; or if there was the same absolute increase of 10% from 50% on Pre-training to 60% on Post-training, the percentage improvement would be 20%. Thus this measure takes into account the initial level of performance in the pre-training test and represents the percent improvement in relation to that level. Mean and Standard Error Performance Gain for each of the four Language Background × Bilingual Status groups are shown for AO/AV training groups in ao and av tests in Figure F = 8.09, p < 0.01, ηp2 = 0.111. Age also interacted with two other factors: there was an interaction of Age × Tone, F = 15.19, p < 0.001, ηp2 = 0.189, and of Age × Tone × ao/av test, F = 9.54, p < 0.01, ηp2 = 0.128. This set of results is represented in Figure Performance Gain scores were analyzed in an Age × Language Background x Tone Experience × Training Mode between-subject factor × Test Type (ao or av) within-subject factor ANOVA. The only significant main effect was for Age, F = 3.91, p > 0.05, ηp2 = 0.057; Training Mode × Monolingual/Bilingual × ao/av tests, F = 3.76, p>0.05, ηp2 = 0.055 . Given these close to significant interactions and the significant interaction of ao vs. av tests with Age and Tone Language results above, and in order to avoid a Type II error in this first test of the effect of training mode on lexical tone perception, these two approaching significance results were followed up in simple effect tests of Training Mode × Monolingual/Bilingual at each level of the test type, ao tests and av tests. These revealed a non-significant Training Mode × Monolingual/Bilingual interaction for av tests, F = 0.70, p > 0.1, ηp2 = 0.011, but a significant Monolingual/Bilingual interaction for ao tests, F = 5.19, p < 0.03, ηp2 = 0.074. This set of results is represented in Figure The above Age and Tone Language background results are independent of whether the children were monolingual or bilingual and whether they were trained with AO or with AV stimuli. Turning to Training Mode and Monolingual/Bilingual, the Training Mode and Monolingual/Bilingual interaction, and the Training Mode × Monolingual/Bilingual × ao/av interaction were both very close to significance Training Mode × Monolingual/Bilingual, F1, 6 = 3.91, Correlations, with age partialed out, between the phoneme deletion, word and non-word reading tests with the pre-training, post-training, and performance gain due to training were conducted for the three groups with English as one of their languages .r(62) = 0.50, p < 0.001, phoneme deletion and non-word reading r(62) = 0.59, p < 0.001, and word and non-word reading, r(62) = 0.75, p < 0.001.There were, not surprisingly, correlations between the three language measures –phoneme deletion and word reading r(62) = 0.26, p < 0.05. This indicates that children's phonological awareness, in this case their proficiency on a phoneme deletion task, is positively related to their initial identification of the four Mandarin tones presented in auditory-visual mode.More important are correlations between any of the three language measures and the tone training scores. The only significant correlation of this nature was between phoneme deletion and the pre-training av-test, This study examined the role of tone vs. non-tone language experience, monolingualism vs. bilingualism, and auditory-only vs. auditory-visual training of foreign lexical tone contrasts. The results are summarized under four headings below, followed by discussion of the results.Training was effective: there was a general improvement in performance from pre- to post-training. Training was most effective for 8-year-olds; 6-year-olds showed only limited effects of training. Training was more effective if children had tone language experience, an advantage evident in the 8- but not the 6-year-olds. These effects of age and tone language on training were most clearly indexed by the ao rather than the av pre- and post-training tests.There was a differential effect for the type of training: Monolingual children improved markedly with AV training but not at all with AO training, whereas Bilingual children improved markedly with AO training and to a lesser extent with AV training. However, these effects were only apparent when indexed by ao tests.For children with English as their only, or as one of their, language(s), proficiency on a phoneme deletion task was positively related to Mandarin tone identification in auditory-visual pre-training test trials. As this was before training began, it shows that those children good at manipulating phonemes in (one of) their native language(s) were also good at perceiving what were completely novel phonological elements for the Mono-Eng, the Bi-Eng/Arabic and the Bi-Eng/Thai groups. This advantage did not extend to training, there was no advantage for good phoneme deleters in learning about foreign tones, just in their initial perception of foreign tones.The results bear on a number of issues which are discussed below ahead of a discussion of limitations and suggestions for future research.There are two possible reasons why training was more effective with the older 8-year-olds than the younger 6-year-olds: task difficulty, and reduced sensitivity to foreign sounds. First it may be that the task employed here was demanding in terms of the degree of sustained attention required. For example, while in the procedure used here the pre- and post-training trials were the same, the training trials incorporated variation of both speakers and words. Wang et al.  trained Secondly, irrespective of task difficulty, 6-year-old children may have reduced sensitivity to foreign sounds. Burnham and colleagues  or bilingual experience (Bi-Eng/Thai).In this experiment children were given both ao- and av-test trials pre- and then post-training. The training was either with AO stimuli in one group and AV stimuli in another group. In addition, the stimulus words and speakers were different in the pre- and post-training test phase on the one hand and in the Training trials on the other. Therefore, generalization of training can be indexed in two ways. First, any improvement after training, can be considered generalization because the training and test stimuli differed . In this sense then, any performance gain from pre- to post-training, such as those gains found in this study, can be considered as both learning, and generalization of learning. Second, generalization can be indexed by any performance gain across both the ao- and the av-Participants with tone-language experience (the Bi-Eng/Thai and Mono-Thai groups) benefitted more from training than those with no tone language experience (Bi-Eng/Arabic and Mono-Eng), irrespective of whether the children were monolingual or bilingual. In addition, those with tone language experience  also showed better generalization of training across test type—ao and av. This supports previous findings that tone language experience facilitates adult lexical tone perception  tone language background on the learning of the target tones in Mandarin, i.e., that there was transfer of learning from Thai tones to learning Mandarin tones. Second, the differences between Thai and Mandarin may have played a part in the relatively small performance gains in tone perception here. Further studies in which the background language and target tone language are more similar with respect to their tones, e.g., Thai and Cantonese , may result in more performance gains. More generally, the relative salience of differences between tones within a particular language and the relative difficulty of discriminating tones in one tone system vs. that in another system is largely unknown  mode of training was the most effective for monolingual children, whereas for bilingual children Auditory-Only (AO) training, and, to a lesser extent, AV training resulted in performance gains. The source of this difference is not clear. One possibility is that exposure to a greater range of linguistic differences and devices, as would be the case for bilingual children, allowed them to (i) learn from a range of parameters, including auditory information alone or auditory and visual information combined, and (ii) learn that, even though there is visual information for tone  was positively related to pre-training auditory-visual test trial performance. Although there was no relationship here between reading and tone perception, the results are reminiscent of those of Burnham et al.  who founA number of limitations can be noted.The post-training test implicitly tested for generalization across speakers and words, and, in addition, these trials provided implicit tests of generalization from training mode (be it AO or AV) to test mode, as both ao and av tests were given irrespective of training. The downside of this is that tests between trained and untrained stimuli and voices could not be conducted. It is possible that children, even the younger 6-year-olds, may have performed better on trained than untrained stimuli and voices. This should be remedied in future studies. The upside is that any improvement as a result of training indicated generalization of training. So the performance gains obtained here, while modest, are robust.variability. As discussed above, variability improves the robustness of learned distinctions (Wang et al., A related point concerns A final point on this theme is that for both clusters of results—the age and tone language experience cluster, and the training mode and monolingual/bilingual language experience cluster—the ao-tests were more sensitive measures of improvement than were the av-tests. And, even though auditory and auditory-visual modes differentially affected training outcomes in monolingual and bilingual children, the indexation of such training was still generally better on ao-tests. The reason for this is unclear. In future studies it would seem that ao-tests should be preferred.We included English language tests of phonological awareness here and found a positive relationship between phoneme deletion and pre-training auditory-visual test performance. Future studies should investigate this further by including reading tests across languages, phonological awareness tests across languages, and also tests of morphological awareness (McBride-Chang et al., No specific instructions were given. Children were simply told to pay attention to both the auditory and visual aspects of the speakers as we wished to determine whether children naturally pick up relevant lexical tone cues in an experimental setting. In real-life L2 learning situations such experimentally objective procedures may not be desired; indeed any relevant cue could and should be made available. In this regard, Chen and Massaro  tested MThe Chen and Massaro  results The results of this study show that 8-year-olds and to some extent 6-year-olds can learn to identify Mandarin tones. The training procedure included feedback during practice and training sessions and rewards in training of animated cartoons after three consecutive correct responses. The same stimuli were used in pre- and post-training, but there was considerable variability of training stimuli in terms of speakers and the word contexts, variability that perhaps hindered learning in children of 8- and especially 6-years of age.per se had no effect on tone training. However, the modality of training appeared to be differentially effective: as indexed in ao-test trials, monolingual children improved markedly with Auditory-Visual training stimuli but not at all with Auditory-Only training, whereas bilingual children improved markedly with Auditory-Only training and to a lesser extent with Auditory-Visual training. These results suggest an interesting interaction between language experience and training method that may have important implications for L2 training techniques and facilitate L2 training regimes.Nevertheless, there was successful training of 6- and 8-year-olds to identify non-native lexical tones. The main findings were that tone language experience with Thai facilitated learning to identify Mandarin tones, irrespective of whether the experience was monolingual Thai, or bilingual Thai-English. Monolingual vs. bilingual experience BK and LE-KA experimental design, data collection and analyses, data management, manuscript drafting and revising, approving the manuscript to be published and Agreeing to be accountable for all aspects of the work in this manuscript. AJ, JAS, and DB experimental design, supervision of data collection and data management, manuscripting drafting and revising, approving the manuscript to be published, and agreeing to be accountable for all aspects of the work in this manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Previous studies have shown that sport experts are different from novices in functions and structures of the cerebellar sub-regions and the functional connectivity (FC) associated with the cerebellum, suggesting the role of the cerebellum on motor skill learning (MSL). However, the manipulation of individuals with different motor skills fails to exclude the effects of innate talents. In addition, individuals with higher motor skills often start with the MSL in their young ages. It is still unclear whether the effects regarding the cerebellum would be shown at one’s adult age. The present study was to directly alter individuals’ motor skills to investigate whether MSL (taking learning to play badminton as an example) in adulthood influences resting-state activity in the cerebellum. To this end, young adults without ball training experience were recruited as participants and were assigned randomly into the experimental group and the control group. Participants in the experimental group were asked to attend a badminton training course for 12 weeks, while the control group did not regularly attend any ball sports during this period. Resting-state functional magnetic resonance imaging (fMRI) was recorded before and after the training. Results showed that compared to the control group, the experimental group had smaller amplitude of low-frequency fluctuation (ALFF) in right cerebellar hemispheric VI and left VIII after training. For the experimental group, right hemispheric VIII had a stronger FC with left hemispheric IV-V, cerebellar vermal IX, left middle cingulate gyrus and right hippocampus after training. Taken together, these findings suggested that MSL, at least learning to play badminton in adulthood, reduces resting-state activity in different sub-regions in the cerebellum but increases FC between sub-regions of the cerebellum as well as between sub-regions of the cerebellum and cerebral cortices . The human cerebellum, hidden underneath the occipital and behind the brain stem, is thought to be a heterogeneous structure consisting of the vermis and two hemispheres, and it has been anatomically divided into many sub-regions  . PreviouResearchers found that motor skill learning (MSL) influences cerebellar structures and functions by observing the corresponding structures and functions of sport experts and novices –22. RegaWith regards to FC, three studies found that MSL altered the FC among different sub-regions of the cerebellum and between cerebellum and other cerebral cortices , 23–24. Notably, all of the aforementioned studies adopted cross-sectional design. This design cannot exclude the influence of innate factors on the effects of MSL. For instance, compared to novices, athletes are often thought to have higher talents in sports –28. The The present study aimed to use a longitude design to investigate the effects of MSL (taking learning to play badminton as an example) on the cerebellum in adults. To address this issue, young adults, who have no any ball training experience, were recruited as participants. They were assigned randomly to two different groups, i.e., the experimental group and the control group. Participants in the experimental group were asked to attend to a badminton training course  for 12 weeks, whereas for the control group, participants remained in their common life. Both of the two groups were scanned by fMRI before and after the training. Moreover, cross-sectional studies have suggested that professional ball training including badminton training alters cerebellar structures and functions –20. CompThe present study was interested only in the effects of MSL on resting-state functions and FC associated with the cerebellum. We would use the ALFF analysis to examine the regional properties of the intrinsic neural activity in the cerebellar sub-regions. The voxel-wise FC analysis was then conducted to characterize functional integration. Based on the literatures –16, the Task-state fMRI studies found that continuous skill learning lead to decreased activity in cerebellum –32. And,M ± SD = 24.22 ± 0.83 years, 15 males) were recruited from Tianjin Normal University in return of payments. Half of the participants were assigned to the experimental group , and the other half to the control group . These two groups did not differ in age (p > 0.1) and education. All participants were right-handed as determined by the Edinburgh Handedness Inventory [Thirty-six undergraduate and postgraduate students  images were acquired using an echo-planar imaging (EPI) sequence. Acquisition parameters were as follows: TR = 2000ms, TE = 30ms, field of view = 220mm × 220mm, flip angle = 90°, matrix = 58 × 64, 25 slices/volume, thickness = 5mm, gap = 0.75mm, voxel size = 3.8mm × 3.4mm × 5mm. The scanning direction is from top to bottom. The scanning mode is interval scan, starting from the odd layer. The resting scan lasted 8min 26s for each participant. The structural (T1-weighted) images were acquired using gradient echo sequence. Acquisition parameters were as follows: TR = 1900ms, TE = 2.52ms, field of view = 240mm × 256mm, flip angle = 9°, matrix = 240 × 256, 176 slices, thickness = 1mm, gap = 0.5mm, voxel size = 1mm × 1mm × 1mm. The scanning direction is from right to left. The scanning mode is sequential scan. The T1 imaging scan lasted 4min 10s for each participant. In the scanner, participants were asked to keep awake with their eyes closed, without any motions or thinking.http://rfmri.org/dpabi) [http://www.mathworks.com/). For each participant, raw data were converted into NIFTI format, and the first ten-time points of resting-state fMRI data were discarded, leaving 240 time-points for further analysis. The further preprocessing included slice timing, head-motion correction (the data of those whose head motion > 1.5 mm in any plane and rotation > 1.5° in any direction were discarded), nuisance covariates regression ), spatial normalization by information from unified segmentation  space using the normalization parameter estimated during unified segmentation.), spatial smoothing  = 8 mm × 8 mm × 8 mm Gaussian kernel), and a band-pass filter within 0.01–0.08 Hz.The preprocessing of resting-state fMRI data were performed using a toolbox for data processing and analysis of brain imaging, DPABI V2.1 (Copyright(c) 2014; GNU GENERAL PUBLIC LICENSE; g/dpabi) , implemeThe filtered data were used to perform ALFF analysis in DPABI V2.1. An ALFF map was calculated for each participant and a normalized ALFF map (mALFF) was also obtained for subsequent statistical analysis. According to the automated anatomical labeling (AAL) template , bilaterhttp://www.fil.ion.ucl.ac.uk/spm/) implemented in Matlab R2012a. Test time is a within-subject factor and group is a between-subject factor. The significant threshold was set voxel-level corrected pFWE < 0.05 by Family-Wise Error and cluster size > 10 voxels. For each ROI, the ALFF signals were extracted, if the interaction effect was significant. Notably, the signals were based on the ROIs from the AAL template. Then, the signals were used to perform post-hoc t-tests with IBM SPSS Statistics software . A probability level of p < 0.05 was considered statistically significant.At group-level analysis, a full factorial design was carried out with a focus on the interaction of test time (pre vs. post) × group  within each ROI in SPM8  using Fisher transformation for subsequent statistical analyses.pFWE < 0.05 by Family-Wise Error and cluster size > 10 voxels. For the zFC between each seed region and each of the other brain regions, the signals were extracted, if the interaction effect was significant. Notably, the signals of the seed regions were based on the ROI from the AAL template and the signals of the other brain regions were based on the voxel-level whole brain. Then, the signals were used to perform post-hoc analyses with SPSS 22.0 and the significant threshold level was p < 0.05.At group-level analysis, a full factorial design was carried out with a focus on the interaction of test time (pre vs. post) × group  in SPM8 implemented in Matlab R2012a. Test time is a within-subject factor and group is a between-subject factor. The significant threshold was set voxel-level corrected A significant interaction between test time and group was found in the right cerebellar hemispheric VI and left cerebellar hemispheric VIII. No significant interaction was found in any other ROIs. See p = 0.088). For the control group, there were no significant differences between pre-test and post-test (p > 0.1). The other direction of further analysis showed that at pre-test, there were no significant differences between the two groups (p > 0.1). At post-test, the ALFF was slightly smaller for the experimental group than for the control group (p = 0.078).Regarding the ALFF of right cerebellar hemispheric VI, further analysis showed that for the experimental group, the ALFF was slightly smaller at post-test than at pre-test (ps. > 0.1). The other direction of further analysis showed that at pre-test, there were no significant differences between the experimental group and the control group (p > 0.1). At post-test, the experimental group had smaller ALFF than the control group (p = 0.005). See For the ALFF of left cerebellar hemispheric VIII, further analysis showed that there were no significant differences between pre-test and post-test in either the experimental group or the control group . However, for the control group, those FCs were smaller in the post-test than in the pre-test (ps < 0.05).Further analysis showed that for the experiment group, the FCs in each of abovementioned two brain areas were larger in the post-test than in the pre-test (ps < 0.05). However, at the post-test, the effect regarding FC was reserved (ps < 0.05). See The other direction of the further analysis showed that at the pre-test, the experimental group had smaller FC in in each of abovementioned two brain areas than the control group (ps < 0.05). Therefore, the results indicate that badminton training is effective on influencing FCs, even though the FCs were different in experimental and control groups before the training.Given that the group effect regarding the abovementioned FCs in the post-test might be due to the group effect in the pre-test as a result of significant pre-test group effect, we performed ANCOVAs on each post-test FC of each of the abovementioned two regions using pre-test FCs as a covariate. Results showed that the post-test FC was larger for the experimental group than the control group ; right cerebellar hemispheric IV-V and right brainstem pons: 0.54±0.04 vs. 0.20±0.04; right cerebellar hemispheric VIII and right hippocampus: 0.48±0.04 vs. 0.19±0.04; right cerebellar hemispheric VIII and left middle cingulate gyrus: 0.34±0.04 vs. 0.12±0.04; vermal IX and right cerebellar hemispheric VIII: 0.47±0.05 vs. 0.23±0.05; vermal IX and left brainstem pons: 0.69±0.03 vs. 0.41±0.03, The present study aimed to investigate whether motor skill learning, taking learning to play badminton as an example, in adulthood influenced resting-state activity of hemispheric IV-VI, VIII and vermal VI-IX of the cerebellum and intra-modular FC within cerebellum and inter-modular between cerebellum and cerebral cortices. The results showed decreased ALFF activity in right hemispheric VI and left hemispheric VIII of the cerebellum for the experimental group but not for the control group after training, suggesting the role of learning to play badminton on the resting-state activity of the cerebellum. In addition, the experimental but not the control group exhibited stronger FC between the several sub-regions of cerebellum and some other regions of cerebral cortices or cerebellum after training, e.g., between left cerebellar hemispheric IV-V and right cerebellar hemispheric VIII, between right cerebellar hemispheric VIII and left middle cingulate gyrus, between right cerebellar hemispheric VIII and right hippocampus, between cerebellar vermal IX and right cerebellar hemispheric VIII. These findings suggest that MSL integrates motor and cognition functions between different sub-regions of the cerebellum and between cerebellar sub-regions and cerebral cortices.Previous studies showed that continuous skill learning affected the function of the cerebellum –32. For As mentioned in the introduction, hemispheric VIII is related to sensorimotor –10. ThisThe hemispheric VI in the cerebellum has been demonstrated to be involved in motor coordination in complex movements, such as visually-guided movements  and sequCompared with the above tasks, learning to play badminton in real life is more complicated and it involves various kinds of motor plan and execution and cognition processes, such as, the movements of the body and limbs, hand-eye coordination and body limb coordination. The cerebellar hemispheric VI and VIII might be activated during learning to play badminton. Therefore, learning to play badminton may repeatedly activate the aforementioned sub-regions of the cerebellum, and lead to their low activities in the resting state. The findings suggest that skill learning of playing badminton decreases the spontaneous neural activity in right hemispheric VI and left hemispheric VIII of the cerebellum.Different from the findings in the present study, Di et al. found that badminton athletes had stronger ALFF in vermal VI than novices , howeverFC reflects the temporal correlation of low frequency fluctuations in the resting-state in the different brain regions , 43. BraOn the other hand, Badminton is set to play in a court with certain length and width. During badminton playing, to strike the ball back within the court, players have to intercept the shuttle from their opponents and strike the ball back with their racquets. Playing badminton involve not only motor plan and execution but also spatial perception and mental motor extrapolation (anticipation the future location of a moving shuttle). Right hippocampus is thought to be associated with the processing of spatial information, such as space navigation . FMRI stWe would like to mention limitation of our study and suggest outlines for future research. Firstly, as mentioned above, the duration of MSL may influence the effect of learning on the ALFF of the cerebellum. Future studies may extend the duration of learning to further investigate the related issue. Secondly, the present study did not exclude the influences of some factors, such as motor skills and experiences , physical characters (height and weight) and abilities of memory, learning, and emotional control. According to the results regarding the group effects, these factors or some of them might have led to differential FCs between experimental and control groups in the pre-test. While the ANCOVA analyses suggested the effectiveness of badminton training even though there were some pre-test differences between experimental and control groups, future studies should control for those factors and reduce the group differences in the pre-test to further investigate the related issues. Finally, it is not clear whether the results can generate in other badminton-similar sports  or even badminton-dissimilar sports . Future studies may include another control group, who attend the trainings of these badminton-similar and badminton-dissimilar sports, to further investigate the related issues.In the present study, we observed that compared to the control group, the experimental group had reduced the ALFF in right cerebellar hemispheric VI and left cerebellar hemispheric VIII after training. In addition, the experimental group exhibited stronger FC  after training. These findings indicate that these brain regions may be activated or co-activated during learning to play badminton, which leads to their lower ALFF and stronger FC at resting-state. The findings may contribute to understanding the changes of cerebellar plasticity induced by MSL, at least learning to play badminton in adults.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(ZIP)Click here for additional data file.S4 File(ZIP)Click here for additional data file.S5 File(ZIP)Click here for additional data file.S6 File(ZIP)Click here for additional data file.S7 File(ZIP)Click here for additional data file.


Trypanosoma brucei. The suggestion was based on a time-dependent inhibition of the enzyme by curcumin and a lack of recovery of activity of the enzyme after pre-incubation with curcumin. This study provides clear evidence that curcumin is a reversible, non-competitive inhibitor of rhodesain. In addition, the study also shows that the apparent irreversible inhibition of curcumin is only observed when no thiol-reducing reagent is included in the measuring buffer and insufficient solubilising agent is added to fully dissolve curcumin in the aqueous solution. Thus, the previous observation that curcumin acts as an irreversible inhibitor for rhodesain was based on a misinterpretation of experimental findings.Previously, it was suggested that the natural compound curcumin is an irreversible inhibitor of rhodesain, the major lysosomal cysteine protease of the protozoan parasite  Curcumin is a natural phenol and has been extensively investigated as potential drug candidate for various illnesses and medical conditions . HoweverTrypanosoma brucei, the causative agent of sleeping sickness in humans and nagana disease in livestock [T. brucei, has been recently investigated [Previously, it was shown that curcumin display anti-proliferative activity against the protozoan parasite ivestock . In searstigated . The enzstigated . It was stigated . This costigated ,5. Howevstigated . A 1:4 dstigated . In ordestigated ,5. The rThe activity of rhodesain was determined with the fluorogenic substrate benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin (Z-FR-AMC), a substrate that is cleaved by mammalian and trypanosome cathepsin L cysteine proteases .50 value for the inhibition of rhodesain by curcumin, see below), the inhibition of the activity of rhodesain was linear with respect to time , the aci for curcumin was determined to be 5.5 ± 1.4 μM (n = 3).After establishing that curcumin is a reversible inhibitor of rhodesain (see above and ), kinetii value is equal to the IC50 value [50 value of curcumin for the inhibition of rhodesain was determined next. The compound inhibited the activity of rhodesain in a dose-dependent manner with an IC50 value of 5.6 ± 0.5 μM (n = 3) . This finding confirmed that curcumin is indeed a non-competitive inhibitor of rhodesain.As for a non-competitive inhibitor, the K50 value ; the IC5 (n = 3) . The IC5p = 0.337; Having shown that curcumin is a reversible, non-competitive inhibitor of rhodesain, the question remains why previously a lack of recovery of activity after pre-incubation of the resting enzyme with the compound was found ,5. As thFinally, the question remains as to why a time-dependent inhibition of rhodesain activity by curcumin was recently observed ,5. In th0’: the more negative E0’, the better the reactivation (E0’(cysteine) = −348 mV; E0’(dithiothreitol) = −323 mV; E0’(β-mercaptoethanol) = −207 mV; E0’(glutathione) = −205 mV; E0’(ascorbic acid) = +58 mV [Other antioxidants  were also able to reactivate rhodesain that had been inhibited by curcumin, although at different effectiveness . Cystein= +58 mV ,16,17). T. brucei cathepsin L-like protease) was provide by Professor Conor R. Caffrey, Center for Discovery and Innovation in Parasitic Diseases, Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, CA, USA. Benzyloxycarbonyl-phenylalanyl-arginyl-7-amido-4-methyl coumarin (Z-FR-AMC) was purchased from BIOMOL, Exeter, UK. Curcumin was bought from Sigma-Aldrich, Dorset, UK.Recombinantly expressed and purified rhodesain . Release of free 7-amino-4-methylcoumarin (AMC) was measured at excitation and emission wavelengths of 360 nm and 460 nm in a BIORAD VersaFluor fluorometer, Watford, UK.In order to determine whether the activity of rhodesain was inhibited in a time-dependent manner by curcumin, the enzyme  was incubated in the presence of 6 μM curcumin dissolved in DMSO in measuring buffer plus 5 μM Z-FR-AMC. For a negative control, rhodesain was incubated with the same amount of DMSO (2%). For a positive control, the enzyme was incubated with 0.1 μM of the established irreversible inhibitor CAA0255 ,9.To determine whether the absence of dithiothreitol had an effect on the inhibitory activity of curcumin, rhodesain  was incubated with 6 μM curcumin in measuring buffer plus 5 μM Z-FR-AMC but lacking dithiothreitol. The release of free AMC was recorded every minute over a period of 30 min. Then, different antioxidants  were added in order to determine whether curcumin-inhibited rhodesain could be reactivated. The release of free AMC was recorded every minute for another period of 30 min.i value were graphically determined by Lineweaver–Burk plot and Dixon plot, respectively.The inhibitor type of curcumin for rhodesain was determined by kinetic analysis. Purified rhodesain was incubated with varying concentrations of Z-FR-AMC  and curcumin  in measuring buffer containing 2% DMSO. The final concentration of rhodesain in the assay was 14 ng/mL (0.4 nM). The release of free AMC was recorded as described above every 30 s for 5 min. The velocity of the reaction (relative fluorescence units (RFU)/min) was calculated by linear interpolation of the data. The inhibitor type and the K50), purified rhodesain was assayed with 5 μM Z-FR-AMC in measuring buffer containing different concentration of curcumin (twofold dilutions from 32 μM to 1 μM) and 2% DMSO. Controls contained 2% DMSO alone. The final enzyme concentrations in the assays were 7 ng/mL (0.2 nM). After 10 min incubation at room temperature, the release of free AMC was recorded. IC50 values were determined by linear interpolation according to the method by Huber and Koella [For determination of the half-maximal inhibitory concentration  was pre-incubated with 100 μM curcumin in the presence of 0.1% or 10% DMSO in measuring buffer for 30 min at room temperature. Then, the mixture was diluted 100-fold into measuring buffer containing 5 μM Z-FR-AMC. After 10 min incubation at room temperature, the release of free AMC was recorded. Controls were pre-incubated under the same conditions but in the absence of curcumin.Through enzyme kinetic measurements, it was unequivocally shown that curcumin is a reversible, non-competitive, inhibitor of rhodesain. Additional time course and dilution experiments provided conclusive explanations as to why previously it was mistakenly suggested that curcumin is an irreversible inhibitor of rhodesain. Taken together, this study has once more confirmed that curcumin is a promiscuous molecule that can interact non-specifically with any protein under certain incubation conditions leading to misinterpretation of results.